Smooth muscle is the contractile element in most hollow organs, e.g. the vasculature and gastrointestinal tract and is essential for physiological function. An important mechanism controlling contractile activity in smooth muscle is phosphorylation of myosin. The level of phosphorylated myosin reflects the activities of 2 enzymes: myosin light chain kinase and myosin phosphatase (MP). A key discovery was that MP could be regulated, both inhibition and activation are proposed. The regulation of MP plays a crucial role in normal and abnormal (e.g. hypertension) smooth appreciation of the interactions involving the MP subunits is essential for an understanding of MP function at a molecular level. In Sp4ecific Aim 1 interactions centered on the myosin phosphatase target (MYPT1) subunit will be examined, including: interaction with substrate; interaction between the and C-terminal parts of MYPT1; and interaction with RhoA.GTP. These aims should clarify an important aspect of basic MP mechanism and will address the molecular basis for inhibition of MP activity. Some of the kinases involved in phosphorylation of the inhibitory site on MYPT1 are identified but there are no data on the reverse phosphatase reaction. It is unlikely to involve the endogenous MP phosphatase (PP1c) since this is blocked by interactions of MYPT1. The objectives of Specific Aim 2 are to define those interactions preventing PP1c action and to identify the phosphatase involved in vivo. The experimental protocol to address this important issue will utilize both in-vitro assays and studies with skinner smooth muscle fibers. It is known that the RhoA and cAMP/cGMP pathways have opposing effects. The possibility that this antagonism involves phosphorylation of MYPT1 by cAMP/cGMP-dependent kinases will be evaluated in Specific Aim 3 and compared to a regulatory mechanism involving phosphorylation of RhoA. Several kinases can phosphorylate MOYTI and inhibit MP activity thus raising the possibility that different signal transduction pathways could be implicated. Two endogenous kinases will be investigated in Specific Aim 4, i.e. an 80 kD unidentified kinase and ZIP-like kinase. The above studies will lead to a more detailed understanding of MAP FUNCTION and regulation in smooth muscle and non-muscle cells and will form a basis for appreciation of abnormal function.